Konstrukcija sojeva Saccharomyces cerevisiae za produkciju aminokiselina nalik mikosporinu

master's thesis

• Title (english): Construction of Saccharomyces cerevisiae strains for mycosporine-like amino acids production
• Author: Vanja Jurić
• Mentor: Irina Borodina (mentor)
• Mentor: Ivana Ivančić Baće (sumentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Sunčica Bosak (član povjerenstva)
• Committee member: Tomislav Ivanković (član povjerenstva)
• Committee member: Duje Lisičić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2019-11-15, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Aminokiseline nalik mikosporinu su spojevi poznati po upijanju UV zračenja te su prisutni u brojnim morskim mikroorganizmima koji luče ove sekundarne metabolite da bi se zaštitili u uvjetima stresa izazvanog UV zračenjem. Ovi spojevi tipično apsorbiraju na valnim duljinama od 310 nm do 360 nm. Njihova fotoprotektivna svojstva čine ih obećavajućim kandidatima za farmaceutske i kozmetičke aplikacije. Cilj ovog istraživanja bila je konstrukcija sojeva kvasca Saccharomyces cerevisiae koji će u kromosomima 10 i 11 imati stabilno integrirani heterologni biosintetski put iz cijanobakterije Anabaena variabilis za proizvodnju aminokiselina nalik mikosporinu. Konstruirana su tri integrativna plazmida koji sadržavaju gene za biosintetski put iz A. variabilis uz odgovarajuće konstitutivne promotore i terminatore. Put je ugrađen u haploidne i diploidne sojeve S. cerevisiae te u derivate tih sojeva koji eksprimiraju heterologni put za konzumaciju ksiloze kako bi uz glukozu mogli rasti i na mediju s ksilozom. Ispravnost plazmidnih konstrukata je potvrđena metodom analize kolonija PCR-om te sekvenciranjem. Metodom analize kolonija PCR-om je potvrđena ugradnja vektora u kromosome S. cerevisiae, a metodom tekućinske kromatografije visoke djelotvornosti (HPLC) te masenom spektrometrijom (LC-MS) detektirani su proizvedeni spojevi iz puta sinteze aminokiselina nalik mikosporinu u konstruiranim sojevima.

Abstract (english)

Mycosporine-like amino acids are compounds known for their UV-absorbing properties and are found in numerous marine microorganisms which produce these secondary metabolites as a way of dealing with UV-induced stress. Their absorption maxima typically range from 310 to 360 nm. The aim of this thesis was to construct Saccharomyces cerevisiae strains with heterologous biosynthetic mycosporine-like amino acids pathway from cyanobacterium Anabaena variabilis stably integrated into chromosomes 10 and 11. Three plasmids containing the pathway from A. variabilis with corresponding constitutive promoters and terminators were constructed. The pathway was integrated into haploid and diploid S. cerevisiae strains and into their derivatives which express a heterologous pathway for xylose consumption so that the strains would be able to grow on medium supplemented with xylose, in addition to glucose. Plasmid constructs were verified by colony PCR and sequencing. Colony PCR was used to confirm vector integration into S. cerevisiae chromosomes and high performance liquid chromatography (HPLC) combined with mass spectrometry (LC-MS) was used to detect the produced compounds from the mycosporine-like amino acids pathway in constructed yeast strains.

• Keywords: aminokiseline nalik mikosporinu; put ksiloza izomeraze; put pentoza fosfata
• Keywords (english): mycosporine-like amino acids; xylose isomerase pathway; pentose phosphate pathway
• Language: croatian
• URN:NBN: urn:nbn:hr:217:565236
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2021-11-15
• Created on: 2020-06-09 11:12:04